Extracellular fluid volume in vertebrates is regulated primarily by the mineralocorticoid hormone, aldosterone, which stimulates Na+ absorption in tight epithelia, particularly the kidney collecting duct. This process plays a pivotal role in the control of blood pressure, and its dysregulation, either due to excess hormone or to target cell defects, is an important cause of hypertension. Although aldosterone is known to act through intracellular receptors to regulate gene transcription, the mechanistic basis of its effects on Na+ transport remains poorly understood. The earliest effect of aldosterone is to stimulate apical membrane ENaC insertion and/or increase ENaC open probability without altering ENaC subunit gene transcription. Recently, significant progress was made in understanding the molecular basis of aldosterone-regulated Na+ transport with the discovery that serum and glucocorticoid-regulated kinase (SGK) is an aldosterone-induced ENaC modulator. SGK mRNA and protein are rapidly increased by aldosterone in kidney collecting duct and wild type SGK stimulates ENaC-mediated Na+ current more than seven-fold in Xenopus oocytes. Interestingly, an SGK mutant that cannot bind ATP ("kinase-dead mutant") not only fails to increase Na+ current but actually decreases it. These opposite functional effects of wild type and mutant SGK are correlated with striking opposite effects on ENaC plasma membrane localization in oocytes. Moreover, antagonism of the phosphatidylinositol-3-kinase (PI3K) signaling pathway has an effect on SGK activity and ENaC-mediated Na+ transport similar to the kinase-dead mutant. These findings strongly implicate SGK as an aldosterone-induced regulator of ENaC activity. However, it remains unknown how SGK modulates ENaC activity in CD cells and what role P13K plays in this process. With these observations in mind, the major goals of the work described in this proposal are to: (1) Determine if SGK is necessary and/or sufficient to mediate the effects of mineralocorticoids on Na+ transport in cultured CD cells. We will express wild type SGK and the kinase-dead mutant in cultured CD cells under the control of a heterologous promoter so that their levels can be controlled independently of mineralocorticoids and their selective effects on Na+ transport examined. We will also determine if mineralocorticoid-stimulated Na+ transport in collecting duct cells is delayed or blocked by disruption of SGK expression or activity. (2) Examine the role of ENaC phosphorylation and apical membrane relocalization in SGK- stimulated Na+ transport. We will determine if SGK directly interacts with and/or phosphorylates ENaC subunits. We will determine if SGK stimulates ENaC localization to the apical membrane of cultured cells. We will use mutagenesis to examine SGK structure-function relationships in oocytes and cultured cells. (3) Examine the role of PI3K in activating SGK and in mediating the effects of mineralocorticoids on Na+ transport. We will examine the effect on ENaC-mediated Na+ transport of blocking PI3K activity. We will determine if constitutively active mutants of SGK, or downstream products of PI3K, overcome the effect of PI3K inhibition on Na+ transport. Together, these studies promise to lead to a greater understanding of the mechanistic basis of blood pressure regulation and potentially to new avenues of treatment for salt sensitive hypertension.